The Regulation Mechanism Of C3 Protein Encoded By Tobacco Curly Shoot Virus On Viral Replication

Geminiviruses are a group of single-stranded circular DNA viruses that infect a variety of plant species and cause substantial economic losses each year in many countries and regions of the world.They have single-stranded circular DNA genomes.They encode six to eight proteins in both virion-and complementary-sense orientations that are involved in viral DNA replication,transcription,encapsidation,movement in the host plant,and insect transmission.Geminiviruses replicate ss DNA genomes via a double-strand DNA intermediate through a rolling-circle replication(RCR)mechanism in the nuclei of infected plant cells using host DNA polymerases and transcription factors.Various studies have shown that viral proteins,the replication-associated protein(Rep,also known as AC1,AL1,or C1),and the replication enhancer protein(REn,AC3,AL3 or C3)are required for viral replication,and C1 protein is essential.At present,there are few studies on the role of geminiviruses encoded C3 proteins in viral replication.Although it has been reported that C3 proteins enhance the replication of geminivirus DNA in the host,it remains to be studied to the role of C3 protein in viral replication,and the effects of host factor expression on viral infection.The monopartite begomovirus tobacco curly shoot virus(TbCSV)is a very important plant pathogen that causes tobacco leaf curl disease.In this study,a TbCSV C3 mutant was constructed,and the potato virus x(PVX)system was used to mediate gene expression of Nicotiana benthamiana.Agrobacterium-mediated genetic transformation,high-throughput sequencing,and bioinformatics analysis were adopted to study the effect of TbCSV encoded C3 protein on the viral replication,host phenotype,and functional details of C3 protein mediated some host factors and their effects on TbCSV infection,and the interaction between the C3 protein and the host factors.All research results of this study are summarized as follows:1.Effects of the TbCSV C3 mutant on viral replicationTo determine the response of the C3 protein to TbCSV symptom formation and viral replication,a TbCSV C3 mutant infectious clone(TbCSV?C3),that contains two start codon mutations that abrogated C3 ORF expression,but did not alter the amino acid sequence of the C2 ORF,was infiltrated into N.benthamiana plants by agrobacterium-mediated inoculation.N.benthamiana systemic leaves infected with TbCSVWT showed obviously visible curling,whereas leaves infected with TbCSV?C3 showed no curling at 5 dpi.Subsequently,systemic symptoms of leaves infected with of both TbCSVWT and TbCSV?C3became progressively more severe,but there was no obvious difference between TbCSV?C3and TbCSVWT inoculated plants after 15 dpi.N.benthamiana plants infected with TbCSV?C3 showed typical leaf curling and vein swelling symptom as well as TbCSVWT at15 dpi,indicating that C3 protein had no effect on viral infection.The positive infection rate in the upper leaves of the twenty agroinfiltrated plants was comparable in TbCSV?C3and TbCSVWT infiltrated N.benthamiana at both 5 dpi and 15 dpi.q PCR analyses revealed significantly reduced DNA accumulation in TbCSV?C3 infected leaves compared to those of TbCSVWT infected leaves at 5-,10-and 15 dpi time course.However,there was no significant difference in the level of DNA accumulation between TbCSV?C3 and TbCSVWT infected plants in the later stage of infection(20 dpi).All above results indicated that the C3 mutation reduced the replication of TbCSV DNA and delayed symptom of N.benthamiana at the early stage of infection(5 dpi).The genomic sequence alignment of C3 ORF sequences of TbCSV?C3 infected N.benthamiana plants showed that the two potential start codon mutations at the 2nd and 122 nd bases had not been altered,thus excluding possible reversion of the mutations.2.Effect of overexpression of TbCSV C3 on viral infection and replicationTo clarify the effect of overexpression of C3 protein on TbCSV infection,the PVX system expression vector PVX-C3 and the transient expression vector p CV-C3 were constructed,and transformed into agrobacterium.C3 overexpressed transgenic N.benthamiana plants were prepared and challenged with TbCSVWT and TbCSV?C3.At 7 dpi,PVX-mediated C3 protein expression enhanced the mosaic symptoms of PVX and also promoted the accumulation levels of PVX in N.benthamiana plants,and also enhanced the accumulation of TbCSV DNA in N.benthamiana.The q PCR data revealed that overexpression of C3 significantly enhanced viral accumulation of TbCSV in agroinfiltrated leaves of N.benthamiana.Both TbCSVWT and TbCSV?C3 DNA accumulation levels were significantly increased in the C3 overexpressed leaves of N.benthamiana compared with wild type at 48 hpi.Similarly,accumulation of viral DNA was significantly reduced in TbCSV?C3 than TbCSVWT infiltrated leaves at 48 hpi.An significant increase was observed in the expression level of the C1,C4,V1,and V2 genes in C3 overexpressed N.benthamiana leaves at 48 hpi.We also detected lower levels expression of these genes in TbCSV?C3 infected leaves compared to TbCSVWT.Those data indicated that the overexpression of C3 protein promoted the accumulation level of viral DNA and the expression of viral genes of TbCSV in inoculated leaves of N.benthamiana.The C3 overexpressed transgenic N.benthamiana were screened for positive plants,and there was no significant phenotypic difference between the C3 overexpressed transgenic and wild type N.benthamiana plants.At 7 dpi and 14 dpi,the DNA accumulation levels of TbCSVWT and TbCSV?C3 in transgenic N.benthamiana were significantly higher than those of wild-type N.benthamiana plants.Those all above results indicated that the overexpression of C3 protein didn't affect the phenotype of N.benthamiana,but promoted the accumulation of TbCSV in N.benthamiana plants.3.Screening of host factors regulated by C3 proteinTo further investigate the host factors regulated by the C3 protein and their functional details in the interaction of C3 protein in the host,N.benthamiana plants were inoculated with TbCSVWT and TbCSV?C3 separately and were conducted for transcriptome sequencing.43370 and 43239 genes were identified in the TbCSVWT and TbCSV?C3 infected N.benthamiana plants,respectively.Among them,40,353(67.46%)and 40,220(67.24%)genes were known genes,and 3017 and 3019 were novel genes,respectively.Of all the identified genes,twenty-three genes were significantly differentially expressed,of which sixteen genes were down-regulated and seven genes were up-regulated.These twenty-three differentially expressed genes were enriched to fifteen GO terms,of which 16 up-regulated genes were significantly enriched to fifteen GO terms,and nine down-regulated genes were enriched to nine GO terms.KEGG enrichment analysis results showed that these differentially expressed genes were enriched in sulfur metabolism,circadian rhythm,host disease resistance,superoxide enzyme,ubiquitination-mediated protein degradation,plant-pathogen interaction,and microbial metabolism.Fourteen genes were randomly selected for verification by q RT-PCR.The results showed that twelve of them had transcriptome sequencing results consistent with q RT-PCR results,indicating that the transcriptome sequencing results were accurate.In addition,the NbNAC2 gene was differentially expressed in TbCSVWT and TbCSV?C3 infected N.benthamiana plants,indicating that NbNAC2 gene was regulated by the C3 protein.4.Effect of NbNAC2 expression on TbCSV infectionTo further clarify the relationship between NbNAC2 and C3 protein,and the effect of NbNAC2 expression on TbCSV infection,the expression level of NbNAC2 in different tissues was analyzed by q RT-PCR.The results showed that the expression level of NbNAC2 was highest in the leaves of N.benthamiana,followed by that in flowers,and the accumulation in roots and stems was relatively lower.Construction of C3 and NbNAC2 proteins by thier C-terminally fused GFP fluorescent protein and transformed to agrobacterium,using the GFP empty vector as a control.N.benthamiana leaves were inoculated by agrobacterium infiltration,and the green fluorescence expression was observed under a laser confocal microscope at 48 hpi.Confocal microscopy results showed that both NbNAC2 and C3 proteins were mainly localized to the nuclei,respectively.C3 and NbNAC2 yeast expression vectors were constructed,respectively,and AD-C3 and BK-NbNAC2 were co-transformed into yeast strains.The interaction of NbNAC2 and C3 proteins was confirmed using the Yeast two-hybrid(YTH)system.NbNAC2 and C3 genes were cloned into a fluorescent expression vector containing the N-terminus or C-terminus of the YFP protein,and inoculated with agrobacterium and observed using a confocal microscope.The Bimolecular fluorescence complementary(Bi FC)results showed that NbNAC2 and C3 proteins interacted with each other in N.benthamiana cells,and DAPI staining results showed that the interaction sites were located in the nuclei.The expression level of NbNAC2 in TbCSV infected N.benthamiana,and in C3 overexpressed transgenic N.benthamiana plants wes analyzed by q RT-PCR.The analysis results of the expression level of NbNAC2 in the TbCSVWT and TbCSV?C3 infected N.benthamiana at different time course showed that the expression level of NbNAC2 was lower in the TbCSVWT infiltrated N.benthamiana plants than that of non-infiltrated plants.However,there was comparable expression level of NbNAC2 in TbCSV?C3 infiltrated and non-infiltrated N.benthamiana plants.At 10 dpi,NbNAC2 in TbCSV?C3 infiltrated plants was lower than that of TbCSVWT,but both TbCSV?C3 and TbCSVWT were higher than that of non-infiltrated plants.At 15 dpi,NbNAC2 in TbCSV?C3 infiltrated plants was comparable to the non-infiltrated plants.,and both were significantly lower than that of TbCSVWT infiltrated plants.Moreover,NbNAC2 in C3 overexpressed transgenic N.benthamiana plants was significantly higher than that of wild type N.benthamiana,suggesting that the expression of NbNAC2 was affected by TbCSV infection and the regulated by C3 protein.In order to further analyze the effect of NbNAC2 expression on the growth and development of N.benthamiana and TbCSV infection,the NbNAC2 gene silencing vector and the overexpression vector were separately constructed,and transformed into Agrobacterium strain,and infiltrated into N.benthamiana,followed by inoculation of TbCSV.The results shwed that PVX-mediated overexpression of NbNAC2 caused obvious leaf roll-up symptoms on the new leaves of N.benthamiana at 7 dpi.The infiltrated N.benthamiana plants showed necrosis symptoms processively at 14 dpi.PVX-mediated NbNAC2 expression was up-regulated by approximately 1,000 folds.In the NbNAC2 transiently overexpressed N.benthamiana leaves,the accumulation level of TbCSVWT was higher than that in the control group,but there was no significant difference in the TbCSV?C3 and that in the control group.TRV-mediated NbNAC2 gene silencing had no significant effect on N.benthamiana phenotype.Challenge with TbCSV inoculation showed that the accumulation level of TbCSVWT was decreased in NbNAC2 gene silenced N.benthamiana.These results indicated that overexpression of the NbNAC2 gene promoted the accumulation of TbCSVWT,but it didn't promote the accumulation of TbCSV?C3.Silencing of the NbNAC2 gene inhibited the accumulation of TbCSV in N.benthamiana plants.All in all,this study found that the C3 protein encoded by TbCSV promoted the accumulation of TbCSV in N.benthamiana and the expression of viral genes,and delayed the development of symptoms of early viral infection.We olso found that C3 can interacted with NbNAC2 and regulated the expression of NbNAC2,thereby affecting the accumulation of TbCSV.All these results help provide scientific evidence for the mechanism of action of C3 protein on geminivirus viral replication.